We have studied innate immune cells and genetic determinates of innate immune responses in HCV infection. Project 1: Responses of innate immune cells. Although one HCV virion may suffice to establish HCV transmission and viremia less than 1% of healthcare workers who are accidentally exposed to low amounts of HCV develop high-level systemic viremia. This may be due to either absence of HCV transmission or to early immune responses that rapidly contain and clear small amounts of transmitted HCV. Here, we demonstrate that even a brief exposure to HCV that did not result in systemic viremia triggered responses innate immune responses. Twelve healthcare workers with accidental percutaneous exposure to HCV-infected blood were prospectively studied after accidental HCV exposure via needlestick for phenotype and function of natural killer T (NKT) and natural killer (NK) cells, kinetics of serum chemokines, and vigor and specificity of HCV-specific T cell responses. Eleven healthcare workers tested negative for HCV RNA and HCV-antibodies. All but one of these aviremic cases displayed NKT cell activation, increased serum chemokines levels, and NK cell responses with increased CD122, NKp44, NKp46 and NKG2A expression, cytotoxicity (as determined by TRAIL and CD107a expression) and IFN-gamma production. This multifunctional NK cell response appeared a month earlier than in the one health care worker who developed high-level viremia, and it differed from the impaired IFN-gamma production, which is typical for NK cells in chronic HCV infection. The magnitude of NKT cell activation and NK cell cytotoxicity correlated with the magnitude of the subsequent HCV-specific T cell response. T cell responses targeted nonstructural HCV sequences that require translation of viral RNA, which suggests that transient or locally contained HCV replication occurred without detectable systemic viremia. Collectively, these results demonstrate that exposure to small amounts of HCV induces innate immune responses, which correlate with the subsequent HCV-specific T cell response and may contribute to antiviral immunity. Project 2: Genetic determinants of innate immune responses. Recent immunogenetic studies reported that single nucleotide polymorphisms (SNPs) near the type III interferon gene IFNL3 are strong predictive markers for spontaneous and treatment-induced HCV clearance 6-8. By RNA sequencing in primary human hepatocytes activated with synthetic double-stranded RNA to mimic HCV infection our collaborator Dr. Prokunina-Olsson, NCI identified an upstream genetic variant in linkage disequilibrium with the previously described SNPs. This variant creates a frameshift and a new IFN gene (IFNL4). It is strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians. Transient overexpression of IFNL4 in a hepatoma cell line induced STAT1 and STAT2 phosphorylation and the expression of interferon-stimulated genes. As part of this collaboration we demonstrated that HCV infection induces IFNL4 in primary human hepatocytes and that transient overexpression of IFN-&#61548;ambda 4 suppresses HCV replication. These findings provide new insights into the genetic regulation of HCV clearance and its clinical management.